Abstract

The discovery of the ubiquitous occurrence of antimicrobial peptides (AMPs) and their role as major organism defense effectors was greatly motivated by the emergence and spread of resistance to antibiotics, while their potential use in fighting resistance has been stimulating AMPs research for over two decades. AMPs constitute the first barrier of defense against pathogen dissemination in pluricellular organisms. As components of the innate immunity, they are able to act on a wide variety of pathogens, such as bacteria, fungi, Protozoa and so on. Antimicrobial peptides from the skin of A. loloensis were studied and analysised in this study.Being stimulated by anhydrous ether for 1-2 min, frog skin surface was seen to exude copious secretions. Skin secretions were collected by washing the dorsal region of each frog with 0.1 M NaCl solution containing 0.01 M EDTA. A new antimicrobial peptide has been isolated from the skin secretion of A. loloensis by gel filtration chromatography and high-performance liquid chromatography. The complete peptide sequencing was NILSSIVNGINRALSFFQ which was undertaken by Edman degradation. Skin cDNA library of A. loloensis was constructed.The cDNA synthesized by SMARTTM techniques was used as template for PCR to screen the cDNAs encoding AMPs. Two oligonucleotide primers, S1 (5’-CCAAA(G/C)ATGTTCACC(T/A)TGAAGAAA(T/C)-3’) is a specific primer designed according to the signal peptide sequences of antimicrobial peptides from ranid frogs.The primer (5’-ATTCTAGAGGCCGAGGCGGCCGACATG-3’) in the antisense direction were used in PCR reactions. Many nucleotide sequences of antimicrobial pepetides from the cDNA library of A. loloensis skin have a special N-terminal primer. These homologies have the open reading frame length of about 400 bp. Finally,we got the full-length sequence of the antimicrobial pepetide,named amolopin-pl.The precursors of amolopin-pl are composed of 62 amino acid residues including predicted signal peptides, acidic propieces, and mature antimicrobial peptides. The preproregion of amolopin precursor comprises a hydrophobic signal peptide of 22 residues followed by an 18 residue acidic propiece which terminates by a typical prohormone processing signal Lys-Arg. The molecular weights of this amolopin is 1921.10 Da, and the pI is 9.75. The preproregions of precursors are very similar to other amphibian antimicrobial peptide precursors but the mature amolopins are different from other antimicrobial peptide families. Among the tested microorganisms, native and synthetic peptides only showed antimicrobial activities against Staphylococcus aureus ATCC2592 and Bacillus pumilus, no effects on other microorganisms. The CD spectroscopy showed that it adopted a structure of random combined withβ-sheet in water, Tris-HCl or Tris-HCl-SDS.Fifiteen different mature antimicrobial peptides of amolopin P were deduced from the cDNA sequences (GenBank Accession Numbers:EU311540-EU311554). More transitions from the base caused mutation of their precursor sequence, signal peptide, mature peptide, spacer peptide mutations. To synonymous and nonsynonymous analysis, the results show that the four regions of synonymous is relatively low, more performance for the nonsynonymous. The remarkable similarity of preproregions of precursors that give rise to very different antimicrobial peptides in distantly related frog species suggests that the corresponding genes form a multigene family originating from a common ancestor.